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Paterson, T.E., Beal, S.N., Santocildes-Romero, M.E. et al. (3 more authors) (2017) Selective laser melting–enabled electrospinning: Introducing complexity within electrospunmembranes. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 231 (6). pp. 565-574. ISSN 0954-4119
https://doi.org/10.1177/0954411917690182
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Selective Laser Melting-Enabled Electrospinning: Introducing Complexity within
Electrospun Membranes
Thomas E. Paterson 1 , Selina N. Beal 1, Martin E. Santocildes-Romero 1, Alfred T.
Sidambe1, Paul V. Hatton 1 *, Ilida Ortega Asencio 1**
*Corresponding author for biocompatibility determination.
**Corresponding author for microfabrication.
1 Bioengineering and Health Technologies Group, The School of Clinical Dentistry,
University of Sheffield, Sheffield S10 2TA, UK.
Abstract
Additive manufacturing technologies enable the creation of very precise and well-defined
structures that can mimic hierarchical features of natural tissues. In this paper we describe
the development of a manufacturing technology platform to produce innovative
biodegradable membranes that are enhanced with controlled microenvironments produced
via a combination of selective laser melting techniques and conventional electrospinning.
This work underpins the manufacture of a new generation of biomaterial devices that have
significant potential for use as both basic research tools and components of therapeutic
implants. The membranes were successfully manufactured and a total of 3
microenvironment designs (niches) were chosen for thorough characterisation. Scanning
electron microscopy analysis demonstrated differences in fibre diameters within different
areas of the niche structures as well as differences in fibre density. We also showed the
potential of using the microfabricated membranes for supporting mesenchymal stromal cell
(MSC) culture and proliferation. We demonstrated that MSCs grow and populate the
membranes penetrating within the niche-like structures. These findings demonstrate the
creation of a very versatile tool that can be used in a variety of tissue regeneration
applications including bone healing.
Keywords
Additive Manufacturing, Selective laser melting, Electrospinning, Stem Cell Niche, Bone
Healing
Introduction
During the past decade there has been a marked growth in the use of additive
manufacturing technologies (AM) for medical and dental applications. AM offers great
possibilities in terms of product development, control and prototype design from which the
medical industry can definitely benefit [1-3]. Apart from a custom design, AM also provides
clear advantages including the opportunity for developing internal complexity, a significant
reduction in waste materials and great possibilities for scaling-up [4].
Selective laser sintering (SLS), Stereolithography (SLA) and Fused Deposition Modeling
(FDM) are three of the most commonly used techniques for the manufacture of scaffolds and
biomaterial devices for regenerative medicine [1]. Although they are all based in the same
principle, “layer by layer manufacturing”, the fundamental science behind each technique is
very different and is intimately related to the availability of materials that can be structured
and ultimately transformed into a medical device. Thus, in general terms, we could say that
stereolithography and related techniques (including two-photon polymerisation) require the
presence of a photocurable working solution [5, 6], selective laser sintering requires a
material in a powder form [7, 8], and fused deposition modelling relies on working materials
that can be extruded via temperature control [9, 10]. The possibilities offered by these
techniques themselves are vast, although their potential can be enhanced further when
combined with conventional techniques historically used for biomedical applications (e.g.
electrospinning). Electrospinning is a versatile fabrication process that uses a high voltage
between a syringe containing a polymer solution and an earthed collector in order to create
3D fibrous constructs. Controlling the processing conditions and the design of the collector is
possible to manufacture complex membranes with different fibre alignment, diameter,
porosity, and topography. The incorporation of additive manufactured collectors within the
electrospinning process permits the creation of intricate structures with micron accuracy.
This combination of techniques was patented in 2013 [11] and it was initially developed
using microstereolithography for emulating aspects of the limbal stem cell niche in the
cornea [12-14]. In essence, the continuous deposition of electrospun fibres on top of a 3D
collector fabricated using additive manufacturing will result in the generation of mats that
reproduce the underlying topography of the 3D collector. This way it is possible to provide
the scaffold with levels of complexity that the electrospun fibres on their own cannot achieve.
There is an increasing interest in designing biomedical devices with the ability of directing
cell behaviour via the inclusion of controlled and intricate topography. In the past 5 to 10
years, the design and fabrication of scaffolds containing well-defined microfeatures has been
identified as a rapidly evolving field, and this growing body of research has shown that
topography can be controlled using a wide range of fabrication routes including laser-based
techniques [15-18], micromoulding [19, 20], compression-based techniques [21, 22] and
electrospinning [12-14]. One powerful approach to create intricate topographies is the
development of ‘synthetic niches’ that are designed to mimic specific aspects of the stem cell native niche. Stem cell niches can be described as complex and well-defined
microenvironments that play a fundamental role in tissue repair, controlling to a certain
extent stem cell renewal and differentiation [23]. The ability to recapitulate aspects of the
stem cell microenvironment opens the door to a broad range of applications in regenerative
medicine, especially in the design of smart biomaterial devices for tissue healing [24, 25].
There is a need to develop innovative biomaterial devices able to be delivered to the patient
in conditions that mimic the physiological environment as closely as possible. The inclusion
of the concept of “Stem Cell Niche” within their design philosophy would be an innovative
and valuable approach in which additive manufacturing technologies play a key role,
allowing the creation of custom devices presenting tailored features with dimensions in the
scale of microns.
In this study, electrospinning has been combined with selective laser melting (SLM) to
produce complex scaffolds in which to study and control cell behaviour. The use of SLM
offers specific advantages. Firstly, the manufactured SLM-metallic collectors can be reused
indefinitely, due to their durability, which can have a tremendous impact in the price of future
medical devices. Secondly, SLM offers great accuracy and it is already used in a variety of
medical and dental applications [26]. Finally, the commercially available rig we have used
allows the fabrication of between 10-15 collectors at once, making the process very efficient.
We describe here the manufacture of a range of electrospun membranes incorporating
different morphologies and distributions of synthetic microenvironments setting the basis for
the design and future delivery of a new generation of devices with enhanced regenerative
capability to be used in bone healing applications. These prototype membranes have been
evaluated using mesenchymal stromal cells (MSCs) to determine biocompatibility and
potential for control of stem cell populations.
Materials and Methods
1. Scaffold Fabrication and Characterisation
1.1. Manufacturing of SLM Collectors
The microfabricated electrospinning collectors were manufactured using a Renishaw SLM
125 machine and Stainless steel 316L. The plates were manufactured using a range of
niche-like morphologies designed using CAD software (Solidworks). The specific processing
parameters for the fabrication of the collectors were: Laser power 200W, Speed 480 mm/s,
Point distance 50 micrometres, and exposure time 70 microseconds. The metallic collectors
were then used as targets in a conventional electrospinning set-up for creating electrospun
positive imprints (see Figure1).
1.2. Manufacturing of Microfabricated Electrospun Membranes
Electrospun membranes were fabricated using solutions of 10 wt% poly(caprolactone) (PCL)
(Average Mw 80000; Sigma Aldrich, UK) prepared in a blend of dichloromethane (DCM)
(Fisher Scientific, UK) and dimethylformamide (DMF) (Fisher Scientific, UK) with ratio 90/10
wt% DCM/DMF [27]. For this, PCL was added to the solvents and continuously stirred at
room temperature until the polymer dissolved completely. Electrospinning was performed
using equipment composed of a PHD2000 infuse/withdraw syringe pump (Harvard
Apparatus, UK) and an Alpha IV Brandenburg power source (Brandenburg, UK). Plastic
syringes (1 mL; Becton Dickinson, UK) were used to drive the solutions into 20-gauge blunt
metallic needles (Intertronics, UK). The voltage applied was 17 kV, the flow rate was 2.5
mL/h, and the distance from the tip of the needle to the collector was 21 cm.
1.3. Characterisation of Electrospun Membranes
The topography of the SLM-fabricated collectors was studied using optical microscopy. The
morphology and size of the synthetic electrospun niches was studied in greater detail using
scanning electron microscopy (SEM, Philips X-L 20). Fibre diameter was measured inside
and outside the niche-like areas using ImageJ software and SEM micrographs at a
magnification of x800. Two (2) different samples were analysed and a total of 4 random
areas per sample were examined. A final number of 80 fibres were measured for each of the
cases.
For collecting micro-CT data, electrospun membranes were mounted in plastic straws (0.4
cm in diameter) and placed into the micro-CT machine (SKYSCAN 1272). No filter was used
and pixel size was set at 4.5 µm with a 0.7° rotation step. No averaging was used and a
360° scan rotation was carried out. NRecon software was utilised to construct the image
series using the following settings: 20% beam hardening, 2 smoothing setting, 4 ring artefact
setting and 0 – 0.1 greyscale range was applied. An area of interest was chosen manually to
contain several niche-areas, and then these specific areas were narrowed down.
2. Biological Testing
2.1. Culture of Mesenchymal Stromal Cell
Mesenchymal stromal cells (MSCs) were isolated from the bone marrow of 5-6 weeks old
male Wistar rats following the method described by Maniatopoulos et al. [28]. The femora of
3 animals were dissected in aseptic conditions, cleaned of soft tissues, and immersed in 10
mL of Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, UK) supplemented with 100 units/ml of penicillin (Sigma Aldrich, UK) and 1 mg/ml of streptomycin (Sigma Aldrich,
UK). The ends of the femora were removed and the bone marrows were flushed into 5 mL of
DMEM supplemented with 10 units/ml of penicillin, 0.1 mg/ml streptomycin, 20 mM alanyl-
glutamine (Sigma Aldrich, UK), and 10% v/v foetal calf serum (Biosera, UK). The cells were
then seeded into 75 cm2 culture flasks containing 10 ml of cell culture medium, and were
incubated at 37°C and 5% CO2 for 24 h. The non-adherent cells and debris were then
washed away with fresh cell culture medium. All cell cultures were inspected daily and the
medium was changed every 48 to 72 h. At near confluence, the adherent cells were
removed from culture using 0.05% Trypsin/0.02% ethylenediaminetetraacetic acid (Sigma
Aldrich, UK), pooled into a single population, and seeded for experimentation or stored for
later use.
For cell culture purposes the electrospun microfabricated membranes were cut into
circles/disks (13 mm diameter) and placed in 24 well plates. Test groups used were
microfabricated scaffold with cells, plain sheet of electrospun PCL with cells, electrospun
scaffolds without cells and TCP controls, 5 replicates and n = 3. The individual membranes
were sterilised in 70% methanol and 30% dH2O for 30 minutes before rinsing with PBS three
times. Membranes were finally left submerged in 1 ml of media for 30 minutes before cells
were added.
2.2. Metabolic Activity and Cell Morphology
The metabolic activity of cells on the 3 different niche designs and plain membranes (without
added topography) was measured using PrestoBlue (resazurin-based dye, n=3). Cells were
seeded at a concentration of 50,000 cells per scaffold, and fluorescence measurements
were taken at 1, 7 and 14 days. Cell viability and proliferation in the microfabricated scaffolds
was compared to both 2D-TCP (tissue culture plastic) controls and plain sheets of PCL
electrospun scaffolds (membranes without microfeatures presenting randomly distributed
fibres).
At each time point membranes were moved to a new well plate to prevent contamination of
Prestoblue with any cells growing in the well. Cells were gently washed with PBS and the
PrestoBlue reagent was mixed at a 1:9 ration in media and 700 µl added to each well for 90
minutes. Three aliquots of 200 µl were taken from each sample and fluorescence
measurements were taken using a microplate fluorescence reader FLx 800 Bio-Tek
Instruments using an excitation wavelength of 540 nm and an emission wavelength of 635
nm. After fluorescence measurements the PrestoBlue solution was removed, the cells were
washed with PBS, fresh media was added and they were returned to the incubator.
For fluorescent staining, MSCs were seeded onto the electrospun membranes at a
concentration of 20000 cells per scaffold and stained with Phalloidin-FITC (to label actin
filaments) and DAPI (nucleic acid stain). At day 5 after cell seeding, the membranes were
fixed in 3.7% formaldehyde in PBS for 20 min at room temperature. 0.1% of triton-X100 in
PBS was added to the samples for 20 minutes before rinsing with PBS. Phalloidin-FITC
(1:500) and DAPI (1:1000) was added in PBS for 30 min. Cells were observed using a
confocal scanning microscope (Carl Zeiss LSM510-META, Germany). Images (1024 x 1024
pixels) were obtained using a Zeiss LSM 510Meta inverted confocal microscope and x10/0.3
water dipping objective, with a pixel dwell time of 6.4 µs. Phalloidin-FITC was excited using a
488 nm laser (20% transmission) and emission detected 505 nm. DAPI was excited using an
800 nm laser (12% transmission) and emission detected between 435 and 485 nm. All
image analysis was performed using Zeiss LSM image browser and ImageJ.
2.3. Histology
PCL scaffolds were mounted in tissue freezing medium (Leica) by submersion in liquid
nitrogen. Samples were sectioned to 10 µm thickness using a cryostat (Leica CM1860 UV)
at a controlled temperature of -24 °C. Slides were rinsed gently under tap water to remove
remaining cryostat Optimal cutting temperature (OCT) compound, and then, they were
submerged on a rack in haematoxylin for 60 seconds before being rinsing with a constant
flow of tap water for 5 minutes. Slides were then submerged in Eosin for 5 minutes and then
1 minute in water. Samples were exposed briefly to 70% Industrial Methylated Spirits (IMS),
95% IMS, and left for 30 seconds submerged in 100% IMS to dehydrate the sample.
Samples were finally submerged in xylene before mounting under a coverslip.
2.4. Collagen deposition (Sirius red staining)
Scaffolds were washed in PBS 3 times and fixed in 3.7% formaldehyde. The scaffolds were
then stained using a solution of 0.1% Sirius red in picric acid (Direct Red 80, C.I. 35780,
Sigma-Aldrich) and placed on a rocker for 18 hours. The scaffolds were then washed with
water until no further dye was eluted. For quantitative analysis scaffolds were de-stained in 1
ml of 0.2 M solution of NaOH and methanol (1:1) for 60 minutes on a rocker. Afterwards, 300
µl from each sample were added in triplicate to a 96 well plate and absorbance was
measured at a wavelength of 490 nm using a spectrophotometer plate reader.
2.5. Stem Cell Markers
Samples were washed in PBS and then they were submerged in 0.1% formaldehyde for 15
minutes. CD44 antibody (Anti-rat with fluorochrome Alexa Fluor 647) (1:250) and DAPI
(1:500) in PBS were added to the scaffolds for 1 hour before being washed with PBS.
Images (1024 x 1024 pixels) were obtained using a Zeiss LSM 510Meta inverted confocal
microscope and x10/0.3 water dipping objective, with a pixel dwell time of 6.4 µs. DAPI was
excited using an 780 nm laser (8.1% transmission) and emission detected between 435 and
485 nm. CD44 was excited using a 633 nm laser (51% transmission) and emission detected
between 650 and 710 nm. All image analyses were performed using Zeiss LSM image
browser and ImageJ.
2.6. SEM fixation
Samples were washed with distilled water for 5 minutes then sequentially submerged in the
following solutions of ethanol (prepared in distilled water) for 15 minutes each: 35%, 60%,
80%, 90% and 100%. Hexamethyldisilazane (HDMS) (Sigma-Aldrich) was made up to a 1:1
mixture with ethanol by weight and added to the samples for 1 hour. 100% HDMS solution
was then added to submerge the samples for 5 minutes, twice. Samples were left to air dry
and then gold coated.
3. Statistical Analyses
Statistical analyses were performed on GraphPad Prism software using two-tailed Student T-test, one-way ANOVA, and post-hoc Tukey tests. In all cases, p values <0.05 were considered as statistically significant.
Results
1. Niche and fibre diameter in the microfabricated electrospun mats
SLM allowed the creation of 3 cm x 7 cm rectangular collectors with 1 mm in thickness. The
average niche diameter of the SLM collectors was calculated using optical micrographs and
ImageJ (n=5). The average dimensions were 667 µm ± 85 (Niche1), 1038 µm ± 60 (Niche 2)
and 1168 µm ± 170 (Niche 3). The electrospun replicas were analysed in the same way
showing the following niche diameters: 892 µm ± 76 (Niche 1), 1158 µm ± 32 (Niche 2) and
1287 µm ± 134 (Niche 3) (See Figure 2 for optical microscopy and SEM images).
One-way ANOVA reported statistically significant differences (F (8, 1071) = 3.937, p =
0.0001) between the diameters of fibres belonging to electrospun mats with and without
niches (see table 1 for the average values of fibre diameters). More specifically, post-hoc
Tukey test showed that statistically significant differences between fibre diameters were
found at niche locations 2a and 2b (p < 0.05), 2a and 3a (p<0.0005), and between the
diameter plain mat (without niches) and location 3a (p <0.005) (see schematic of locations in
Figure 3).
It was observed in the SEM images that the density of the fibres per mm2 was visibly lower
in the inner areas of the niche structures (see Figure 2 N-P). This was then corroborated
with Micro-CT scans.
2. Rat MSCs characterisation on microfabricated electrospun mats
Rat MSCs grew and proliferated in the microfabricated scaffolds. MSCs were observed fully
infiltrating the artificial stem cell niches, and confocal z-stacks enabled the accurate imaging
of 3D niche areas (see Figure 4B). Sectioned samples showed cells did not penetrate
greater than 40µm into the fibrous plain mats but they did achieve deeper penetration in
niche areas, as mapped using the DephCod tool in the LSM Confocal software (See Figures
4E and 4F). SEM images corroborated cell attachment (See Figure 4D) and cell distribution
showing that the cellular population of the niches was less dense than in the surrounding not
microfabricated areas (See Figures 4C, 4D).
Discussion
Here we present for the first time the use of selective laser melting for introducing complexity
within electrospun membranes for biomedical applications. The use of SLM for creating
electrospinning collectors is versatile and efficient, as the designs can be easily changed
and adjusted with a high degree of accuracy in terms of size, morphology, depth and
distribution of the incorporated microfeatures. On the other hand, the metallic collectors can
be re-used indefinitely (which is highly desirable from a future end-product point of view).
Electrospun membranes are well-known due to their great potential as regenerative
medicine constructs since they can mimic, to a certain extent, the 3D extracellular matrix,
providing cells with mechanical support and with a porous environment in which to
proliferate. In this work we have achieved the incorporation of a second level of complexity
within our electrospun membranes, which has been introduced via the use of SLM. We have
provided the electrospun constructs with artificial well-defined microenvironments as an
extra tool for influencing cell behaviour. The development of niche-like environments is
indeed a new and rapid growing area of research [12-17, 19, 20, 29, 30]. In this specific
study we have used SLM to aid in the development of electrospun membranes containing
microfeatures and we have chosen 3 types of topography (niche structures) to develop a
preliminary study using primary mesenchymal rat stem cells.
The accuracy in reproducing the features incorporated within the underlying metallic
template was high (higher than 70% for the 3 topographies studied); it was observed that a
decrease in accuracy of reproduction was intimately related with a decrease in the size of
the microfeature. However, this fact does not present a problem for the proposed approach
since we aim to work with features with dimensions ranging from 250µm to 1000µm, which
are biologically relevant in terms of reproducing aspects of a physiological niche
environment. It is also possible to accommodate for this effect when designing the
templates, making the features a different size so that the eventual microfeatures on the
electrospun material are the desired size and dimensions.
Fibre diameter was studied in different parts of the microenvironments (see schematic in
Figure 3) and it was compared to a plain random mat of electrospun fibres. It was observed
that the diameters corresponding to the areas in which the scaffold stretches to reproduce
the morphology of the underlying collector the fibres presented a certain degree of alignment
and, for these cases, the diameter was significantly smaller. These differences are
consistent for all the distinct fibre patterns showed in this study and we attribute the changes
in diameter to the stretching of the fibres during the formation of the scaffold. For example,
for Niche 2, the bottom of the niche (a) presents a random distribution of fibres with a very
similar diameter to the areas outside the niche (c); on the other hand, when we compare the
diameter of the fibres in the bottom of the niche with the diameter on the wall of the niche (b)
we observe a difference in the overall distribution of the fibres (which appear to be more
aligned) and we also observe a significant difference in their diameter. Changes in fibre
diameter can affect cell behaviour, as previously reported in the literature [31, 32]; in our
case, we have performed an accurate study showing that our new methodology allow us to
introduce complexity within the membranes creating niches with different fibre densities and
different areas of fibre diameter which we hypothesise will have a direct effect in influencing
cell behaviour.
Mesenchymal stromal cells were found to attach to our structures and they were located
within the microfeatures (See Figure 4). Confocal Z-stacks provided us with information
regarding the distribution of cells within the niche structures and in their surroundings as well
as regarding the degree of cell penetration within the scaffolds. Cells were homogeneously
distributed within the scaffolds and the use of heat maps was key in allowing us to visualise
the areas in which cells populated the niche structures. Cells were able to proliferate on the
PCL scaffolds as expected; (see PrestoBlue results in Figure 4). Proliferation rate was found
to be slower for the cells seeded on the PCL electrospun mats compared to our Tissue
Culture Plastic (TCP) controls; we anticipated this outcome, since differences in cell
proliferation within 2D (tissue culture plate) and 3D (scaffolds) samples are well documented
in the literature [33]. No significant differences were observed between the different types of
scaffolds with niche morphologies 1-3 or when comparing the scaffolds to a plain mat of
fibres; the inclusion of intricate topography does not seem to have a direct effect in the
degree of cell proliferation which is supported by our previous publications in which
comparable results were reported using equivalent metabolic activity assays [13, 14].
The cells were found to produce collagen, which was measured using Sirius Red (Figure 5),
and no differences between a plain scaffold and the niche-decorated scaffolds were
observed. Collagen production can be used as a guide to determine whether MSC cells
have differentiated into ECM producing cells such as Osteoblasts. The presence of
extracellular collagen can indicate the proportion of cells present that are producing ECM, as
an indicator of osteoblast activity. MSC cells have a rapid proliferation rate and produce
lower quantities of ECM as a result. In contrast, the main function of an osteoblast cell is to
produce ECM and to facilitate the calcification of bone. Further research needs to be carried
out to determine the relationship between the niche-structures and osteoblastic behaviour, in
this preliminary work we just aimed to demonstrate the general osteoblastic capability of our
system.
In this study, CD44 was successfully imaged on the surface of MSCs using confocal
microscopy; CD44 is a surface glycoprotein which is involved in cell adhesion, proliferation,
differentiation and migration processes, and has also been associated with cancer stem cells
[34]. The International Society of Cell Therapy (ISCT) has stated that positively identified
human MSCs must express CD105, CD73 and CD90 [35]. However, the situation for animal
models (a rat model in our case) is not as well defined; for example, CD105, CD73 and
CD90 are not expressed equally by all species [36] and, on the other hand, additional
markers, (such as CD44) are more consistently expressed across species. Nevertheless, the
simultaneous presence of CD44 in many cell types (e.g. MSCs, haematopoietic stem cells,
lymphoid, myeloid, megakaryotic, erythroid and endothelial cellular lineages) reduces its
specificity and limits its use severely [36]. Additionally, evidence has shown that the levels
of CD44 expression in rat bone marrow MSCs may vary significantly depending on animal
strain and passage [37]. In this context, Barzilay et al [37] reported significantly high and
constant levels of expression of CD90 and CD29 antigens on bone marrow MSCs isolated
from four different rat strains and (at passages 2 and 7) which indicates that CD90 and
CD29 would be a good alternative for a more extended study. In our preliminary study the
number of CD44 positive cells seemed to be lower within the niche areas but exhaustive cell
count studies need to be performed to support this claim and, as explained above, the use of
extra markers will be necessary to fully understand the relationship between the niche
environment and the specific behaviour of both a single cell and a cell population. Future
work will focus on the use of CD90 and CD29 in order to enhance the identification of our
MSC cells and determine if cell stemness is directly influenced by the presence of the
microfabricated niche. In this sense, is also known that the incorporation of biomolecules
and/or polymer coatings can also influence and direct cell behaviour [17, 38, 39]; current
work developed in our laboratory is now focussing on the incorporation of specific
biomolecules within our SLM-assisted optimised niche environments; these molecules are
aimed to encourage stemness and we believe will be crucial in dictating the future
regenerative capacity of the overall constructs.
To summarize, in this piece work we have successfully developed and established a
manufacturing method for the fabrication of complex electrospun scaffolds containing niche-
like structures and we have demonstrated the ability of these scaffolds to support MSC
growth and proliferation showing the potential for these membranes to be used in bone
regeneration and related musculoskeletal applications.
Conclusion
This paper reports for the first time a manufacturing method to rapidly and reproducibly
fabricate intricate 3D features within biocompatible electrospun membranes using a metal
template itself produced using SLM. These complex electrospun membranes have the
potential to be used as tissue engineering scaffolds and/or as components of biomaterial
devices with enhanced regenerative capability. The inclusion of SLM within the fabrication
process allows a wide range of possibilities of design and the creation of bespoke tailored
features. Moreover, the metallic collectors used for electrospinning may be reused, so
having a direct impact in future scaling-up procedures and commercialisation of the end
product.
Acknowledgements
The authors would like to acknowledge the Women Academic Returners' Programme
(WARP) at Sheffield for funding part of this work and MeDe Innovation (the UK EPSRC
Centre for Innovative Manufacturing in Medical Devices, grant number EP/K029592/1).
Table Captions
Table 1. Average fibre diameters for each area of the Niche structures. Mean ± SD is
presented in this table.
Figure Captions
Figure 1. Schematic of the manufacturing process comprising two parts: (1) the use of SLM
for manufacturing the stainless steel templates with a variety of niche-like topographies
(fitting 10 templates in a single built) (1); the use of electrospinning for creating a
biodegradable complex electrospun replica with controlled topography (2).
Figure 2. Optical and scanning electron microscopy micrographs of SLM-manufactured
metallic collectors and electrospun scaffolds. Images A-D show a plain collector and three of
the chosen niche-morphologies (x1 magnification); Images E-H are higher resolution optical
micrographs showing the metallic structures (x5 magnification); Images I-L show plain and
niche- electrospun mats (x1 magnification); Images M-P are SEM micrographs of the plain
electrospun scaffold and the three selected niche morphologies, scale bar is 1 mm.
Figure 3. Plot highlighting the differences in fibre diameter between plain scaffolds and Niche
1, Niche 2 and Niche 3, and schematic highlighting the different areas of the Niche
structures chosen for measuring fibre diameter (“a”, inside the niche; “b”, lateral or side of
the niche; “c”, outside the niche).
Figure 4. PrestoBlue values (emission at 635nm) at 1, 7 and 14 days showing the
proliferation of MSC cells both in plain scaffolds and niche morphologies; the samples were
compared to a TCP-2D control. No significant differences were observed between the plain
scaffold and the scaffolds containing microfeatures (A); confocal z-stack of Phalloidin-FITC
stained (green) rat MSCs on a scaffold 2 Niche-like structure (x10 magnification) (B); SEM
image of a type1 Niche-like structure supporting MSC cell growth within the
microenvironment area and in its surroundings (scale bar is 200 µm) (C, D); D’ is showing an SEM false coloured micrograph of an MSC cell inside the niche microenvironment; Confocal
image of a scaffold 2 niche presenting MSC growth (phalloidin-FITC (green) and DAPI
(blue)) within and outside the microenvironment (x10 magnification) (E); Confocal
representation highlighting the differences of depth within a microenvironment type 1 and its
surroundings showing a map of cell nuclei distribution (F).
Figure 5. Sirius red measurements showing collagen deposition in samples with niches and
plain scaffolds. A plain scaffold was used as a control. No significant differences were
observed between scaffolds with and without niches (A). Confocal image of DAPI and CD44
on a plain scaffold after 7 days in culture (x10 magnification) (B). Confocal image of DAPI
and CD44 on a type 1 scaffold after 7 days in culture (C). H&E stain of a 10 µm thick section
of a scaffold (x5 magnification)(D). H&E optical image of a 10 µm thick section of a scaffold
displaying the inter-niche area (x20 magnification) (D’).
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